Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells

Abstract

Licorice root contains chemically diverse compounds that exhibit estrogenic effects in vitro and in vivo. The chalcone isoliquiritigenin (ISL) is a component of licorice extract exhibiting either antitumorigenic activity or estrogen receptor (ER) α-dependent growth promoting effects on breast cancer cells. In order to contribute to a better understanding of this apparent paradox, we synthesized and ascertained the estrogenic properties of ISL using, as model systems, the hormone-sensitive MCF7 breast cancer cells and the steroid-independent HeLa cells.

Transfection experiments reveal that ISL is able to transactivate the endogenous ERα in MCF7 cells and this is supported by the capability to induce down-regulation of ERα protein levels and up-regulation of pS2 mRNA. Moreover, by using chimeric proteins consisting of the hormone binding domains of ERα and ERβ fused to the Gal4 DNA binding domain, we have determined that ISL is an estrogenic agonist of both ER isoforms.

As a biological counterpart, low and intermediate ISL concentrations that induce substantial transcriptional activity stimulate the proliferation of MCF7 cells. However, high levels of ISL become cytotoxic even in steroid-receptor negative HeLa cells. Thus, the activity of ISL and the balance between risk or chemopreventive factor for estrogen-dependent breast cancer may depend on dietary intake.

Introduction

Breast cancer is the most common female malignancy in western countries where the incidence rates have increased steadily over the past decades [1]. In the United States alone, 200,000 new cases are diagnosed every year and 50,000 women die from the disease [2]. A large body of data suggests that estrogens influence both the normal proliferation, differentiation and physiology of breast tissues and the development and progression of breast tumor ([3] and references therein). The effects of 17-β estradiol (E2) and related compounds are mainly mediated by two members of the nuclear receptor superfamily, the estrogen receptor (ER) α and β. Upon ligand binding they undergo a conformational change leading to chromatin interaction and regulation of transcription of target genes [4]. Because estrogens induce the proliferation of many breast tumor cells, this has led to the use of anti-estrogens such as hydroxytamoxifen (OHT) for endocrine therapy. It is worthy to note that the expression of ERα in cancer samples is considered an important prognostic marker associated with higher survival rates and lower risk of relapse [2].

The phytoestrogens, natural compounds abundantly present in soybeans, vegetables and fruit [5], exert in breast cancer cells either growth stimulatory or antiproliferative effects in a dose-dependent manner ([6] and references therein). Interestingly, epidemiological and case–control studies have reported a negative correlation between breast cancer incidence and the urinary excretion of phytoestrogens [7], [8]. Although interpretations of data and conclusions have been contradictory [9], [10], [11], [12], [13], [14], phytoestrogens are considered natural chemopreventive agents [15], [16], [17], [18] that may act through several mechanisms [19], [20], [21], [22], [23], [24].

It has been recently reported that a subclass of the flavonoid compounds isolated from licorice root and named isoflavans are ERα agonists both in vitro and in vivo [25], [26]. Liquorice root was classified in the USA as Generally Regarded As Safe (GRAS) [27] and was listed by the Council of Europe as a natural source of food flavoring in category no. 2 indicating that small quantities can be added to food stuffs to limit the amount of an active compound in the final product [28]. Moreover, some components of licorice root have been shown to exert antioxidant and antimicrobial activities [29], [30], [31] and licorice has been indicated for the treatment of hyperlipidemia, atherosclerosis, viral diseases and allergic inflammation such as hepatitis and atopic dermatitis [32], [33]. An isoflavan and biosynthetic intermediate of flavonoids named isoliquiritigenin (ISL) (Fig. 1) has been recognized as a potent antitumor promoting agent [34], [35] even though stimulatory effects on T-47D breast cancer cells have been well documented [25].

In order to provide new insights into the mode of action of ISL as a natural estrogen-like compound, we synthesized this natural chalcone and ascertained its ability to activate both ERα and ERβ, to modulate ERα target genes, and to induce dose-dependent growth stimulatory or antiproliferative effects in hormone-sensitive MCF7 breast cancer cells as a counterpart of its biological activity.